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Department of Haematology, Royal Free and University College School of Medicine, London, United Kingdom
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(3), and other cytokines, for example, induce EC death (4), while other
growth factors such as fibroblast growth factor and vascular
endothelial growth factor delay apoptosis (5). Direct cell:cell contact can also influence cellular responses, and adherent leukocytes recruited to an area of inflammation may alter EC function and survival. Monocytes are found consistently at sites of inflammation. The recruitment of these cells from peripheral blood is considered to involve sequential adhesive events at the vascular interface (6). There is now increasing evidence that such adhesive events are not only the basis for cell migration, but can also lead to cellular activation, thus playing a role in the regulation of inflammatory responses (7, 8). Monocytes interacting with unstimulated endothelium are induced to express tissue factor, mediated in part by engagement of ICAM-1 (9). Monocytes also induce expression of E-selectin on EC via a cell:cell contact-dependent pathway in which ß2 integrins play a role (10). In a similar manner, monocytes may also influence the integrity and survival of EC.
The balance between cell survival and apoptosis is dependent upon the relative expression of specific genes whose products interact to determine the final outcome of apoptotic signals. Proteins belonging to the bcl-2 family appear to play a central and prominent role in maintaining cell viability (11). bcl-2 is an intracellular protein that blocks apoptosis and prolongs survival (12). EC express bcl-2 at low levels, but also the bcl-2 homologues A1 (13) and bcl-xL (8), which promote cell survival, as well as Bax, which causes accelerated apoptosis. While bcl-2 is up-regulated by growth factors (5), A1 appears to be regulated by inflammatory cytokines (13).
In this study, we have examined the ability of monocytes to influence expression of A1 in EC and investigated whether coculture with monocytes can rescue EC from apoptosis. We have also characterized the cellular interactions responsible for these effects.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelial cells. EC were isolated from human umbilical veins by collagenase treatment and established in culture in Iscove’s modified Dulbecco’s medium (Sigma, Poole, U.K.), 20% FCS (Seralab, Loughborough, U.K.), 20 U/ml heparin, and 50 µg/ml EC growth supplement (Sigma) on fibronectin-coated tissue culture flasks.
Leukocytes. Monocytes were isolated from peripheral blood by starch sedimentation and centrifugation over Nycoprep (S.G.1.068), followed by platelet depletion by centrifugation over autologous plasma, as detailed elsewhere (10). Neutrophils were purified from venous blood using double density centrifugation (Histopaque 1119 and 1077; Sigma), and washed twice in HBSS with 5 mM glucose and 2% FCS. Neutrophils obtained by this method were >95% pure, as assessed by morphology, and >99% viable by trypan blue exclusion. To obtain lymphocytes, mononuclear cells were depleted of monocytes by incubating with anti-CD14-coated Dynabeads for 10 min at room temperature. The resulting lymphocyte suspensions contained less than 1.5% monocytes, as determined by double staining for CD3 and CD14 and analysis by flow cytometry.
All reagents used for cell isolation were screened routinely for endotoxin contamination using the E-Toxate Limulus amebocyte lysate assay (Sigma), and contained less than 0.03 endotoxin U/ml.
Serum starvation of EC
Confluent EC in 25-cm2 flasks or six-well plates were washed with HBSS and then serum starved using 0.1% FCS and a supplement of insulin/transferrin/sodium selenite (RPMI/0.1% FCS/ITS) for 5 h. EC cultures were then coincubated with monocytes, lymphocytes, or neutrophils resuspended in RPMI/0.1% FCS/ITS at 1 x 106 cells/flask (1:1 cell ratio), IL-1 (20 U/ml), TNF (100 U/ml), or medium as control for up to 21 h. In some experiments, monocytes were separated from EC by millipore filters (0.45 µm, Transwell; Costar, Cambridge, MA) to prevent direct cell:cell contact.
Functional inhibition experiments were performed by preincubating monocytes with mAbs against CD11b (M1/70, Boehringer Mannheim, East Sussex, U.K.; 7E3, a gift of Dr. Barry Coller, Stony Brook, NY), CD18 (60.3, gift of Dr. John Harlan, University of Washington, Seattle), VLA-4 (Becton Dickinson, Cowley, U.K.), L-selectin (DREG-56, gift of Dr. Rothlein, Boehringer Ingelheim, CT), or EC with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) (1.3, gift of Dr. P. Newman, Blood Research Institute, Milwaukee, WI) for 5 min at room temperature before use in cocultures. All mAbs were used at 20 µg/ml. In all experiments, an isotype-matched mAb was used as a negative control. In experiments designed to investigate the role of TNF and IL-1 in EC/monocyte cocultures, monocytes were preincubated with anti-TNF mAb, F(ab')2 (gift from Dr. M. Kaul, BASF Pharma, Ludwigshafen, Germany), anti-IL-1 (Peprotech, London, U.K.), or isotype control mAb for 5 min at room temperature, before addition to EC for coculture experiments. All Abs were present throughout the coculture period.
RNA extraction and competitive RT-PCR
Monocytes were removed from EC by incubation with 2 mM EDTA, at 37°C for 3 min, and EC were harvested from the flasks using trypsin/EDTA. The purity of the resulting EC preparation was >99%, as assessed by staining with anti-CD14 mAb for contaminating monocytes using the alkaline phosphatase anti-alkaline phosphatase method. Total RNA was isolated as described (14). One microgram of RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Paisley, Scotland, U.K.) and random hexamer primers (Boehringer Mannheim).
Mimic templates for competitive RT-PCR of A1 cDNA were constructed
essentially as described (15, 16) and consisted of luciferase gene
sequences flanked by sequences complementary to the A1 forward and
reverse primers (Table I
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templates were constructed by PCR amplification of a cloned luciferase
gene (pGL2 vector; Promega, Southampton, U.K.) using tandem
primers consisting of luciferase forward and reverse primers linked to
the 3' ends of the A1 forward and reverse primers, respectively. Mimic
templates for competitive RT-PCR analysis of actin,
bcl-xL, bcl-2, and bax
cDNAs were constructed in a similar manner and are described elsewhere
(16). Competitive RT-PCR analysis (15) of cDNA preparations was
conducted in 25-µl reactions using Taq DNA polymerase
(Life Technologies). Each reaction contained a fixed amount of the
appropriate PCR mimic as an internal standard. The primers used for
amplification of A1 cDNA are detailed in Table I. The reaction was
conducted for 34 cycles using an annealing temperature of 56°C,
yielding a 286-nucleotide product from A1 cDNA and a 470-nucleotide
product from the A1 mimic. PCR products were fractionated by agarose
gel electrophoresis and photographed under UV illumination. Band
intensities were quantified by laser densitometric scanning (Molecular
Dynamics, Sunnyvale, CA; Personal Densitometer) and normalized with
respect to the intensity of the mimic band obtained in each
amplification. The results were expressed as a ratio of the A1 band
intensity relative to the intensity of the actin band obtained by
amplification of the same cDNA (A1/A1 mimic divided by actin/actin
mimic). Data relating to bcl-2,
bcl-xL, or bax band intensities were
treated in an analogous fashion.
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Adherent cells were removed with the use of trypsin/EDTA. Nonadherent and adherent cells were pooled and centrifuged at 1000 rpm for 5 min. Cell viability was assessed using trypan blue exclusion. Cytospin preparations were stained with May-Grunwald-Giemsa. Apoptotic cells were identified by the presence of nuclear condensation and fragmentation (17). All slides were assessed by two observers in a blinded fashion. The remaining cells were used in a FACS-based assay employing dual staining with annexin V-FITC/propidium iodide (PI, Apoptosis Detection Kit; R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Samples were analyzed on a flow cytometer (FACScan; Becton Dickinson).
Statistics
Results are expressed as mean ± SEM. Data were compared using Student’s t test, and a p value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EC were cultured to confluence and rinsed, and the culture medium
was replaced with serum-free medium, as detailed above. After 5 h,
monocytes (1 x 106/flask), IL-1 (20 U/ml), or medium
alone was added to each flask. Following removal of monocytes, EC were
harvested and analyzed for A1 and actin mRNA expression by RT-PCR. EC
grown in complete culture medium (EC -5 h) expressed A1 mRNA, which
was decreased after 5 h of serum starvation (EC +0 h,
p < 0.01, Fig. 1A). EC cocultured with
monocytes for an additional 6 h (EC + MO +6 h) contained more A1
mRNA (A1:actin ratio 0.33 ± 0.03) than did EC cultured alone for
the same period of time (EC +6 h, A1:actin ratio 0.03 ± .01,
p < 0.01, n = 5). Peripheral blood
monocytes do not express A1 mRNA under these conditions (Fig. 1A), and thus could not have contaminated RT-PCR of EC. In
addition, EC harvested for RT-PCR and survival analysis contained less
than 1% contaminating monocytes, as determined by staining with
anti-CD14.
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B). When cDNA from serum-starved EC
cocultured with monocytes (EC + MO +6 h) was tested, the relative
intensity of the A1 amplification product was at least equivalent to or
exceeded that of the actin product when 0.1 or 1 µl of cDNA was used.
When cDNA from control EC (11-h serum starvation in the absence of
monocytes, EC +6 h) was tested, the relative intensities of A1 PCR
products were substantially lower than that of actin products at all
input levels (Fig. 1B). The A1 band intensity became
equivalent to the A1 mimic band intensity at cDNA inputs between 0.1
and 1 µl in the EC + MO +6 h sample. In contrast, A1 bands did not
achieve equivalence with the mimic bands when the EC +6 h sample was
tested, even at cDNA input levels of 1 µl. A similar titration using
cDNA from EC after 5-h serum starvation, i.e., before the addition of
monocytes (EC 0 h), additionally confirmed that A1 mRNA expression
was very low in these cells (data not shown).
Stimulation with IL-1 (EC + IL-1) also increased A1 expression in
serum-starved EC (Fig. 1, A and C). Expression of
A1 in response to IL-1 was short-lived, in that it declined by more
than one-half at 21 h (A1:actin ratio 0.07 ± 0.06 at 21
h, cf 0.23 ± 0.05 at 6 h, p < 0.01,
n = 4, Fig. 1C). In contrast, the
stimulation of A1 expression by monocytes was sustained (Fig. 1C), and levels at 21 h were not significantly lower
than those at 6 h.
Neither neutrophils nor lymphocytes had any significant effect on the
expression of A1 in EC under these conditions (Fig. 2). Monocyte suspensions contained a
variable (5–15%) number of contaminating lymphocytes. Despite
repeated attempts to deplete CD3+ cells, we never achieved
less than a 5% level of contaminating lymphocytes. Therefore, we
cannot discount the possible contribution of a small number of
lymphocytes to the A1 response seen in our EC/monocyte cocultures.
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In one series of experiments, the degree of apoptosis in EC
cultures was assessed by morphologic analysis of cytospin preparations.
Serum starvation increased the percentage of apoptotic EC, from 6
± 1.76% to 28.7 ± 5.8% at 21 h (p
< 0.01, n = 4). When serum-starved EC were cultured in
the presence of monocytes, however, there was a significant decrease in
apoptosis, to 18 ± 3.2% (p < 0.01,
n = 4, Fig. 3A).
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B
shows FACS histograms from one representative experiment. In four
experiments, the percentage of live EC in serum-starved cultures was
62.7 ± 4.8%, which was significantly lower than control EC grown
in complete medium (87.8 ± 3.7%, p < 0.01,
n = 4). Coculture with monocytes increased the
proportion of live EC in serum-starved cultures to 75.8 ± 5.3
(p < 0.05, n = 4, Fig. 3C). Apoptotic cells eventually take up PI because of loss
of integrity of the cell membrane, and thus appear in the
annexin-positive, PI-positive upper right quadrant. Hence, the
percentage of apoptotic EC determined by this method is considerably
lower than that obtained by morphologic analysis. Monocyte stimulation of A1 expression requires cell:cell contact
To determine whether contact between monocytes and EC was needed
to stimulate A1 expression, additional experiments were conducted in
which monocytes were cultured on a 0.45-µm filter above EC. When
contact between monocytes and EC was prevented in this way (EC + MO
filter, Fig. 4A), EC displayed
much lower A1 mRNA levels when compared with EC that had been
cocultured in contact with monocytes (EC + MO, Fig. 4A). In
two separate experiments that were analyzed at 6 h, the levels of
A1 mRNA were reduced by 100 and 57% in EC separated from monocytes,
compared with parallel cultures in which EC were in contact with
monocytes. When A1 expression was determined at 21 h, there was
50.2 ± 7.5% reduction in A1 mRNA levels in EC + MO on
filters, compared with control cocultures
(p < 0.01, n = 4, Fig. 4B). This suggests that contact between monocytes and EC is
required to achieve optimal induction of A1 expression in HUVEC.
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C). EC cultured on
filters showed increased A1 expression when stimulated with IL-1
(A1:actin ratio 0.11 at 21 h), confirming that culture on filters
per se did not alter the ability of the cells to respond to agonists.
We conclude therefore that monocytes in contact with EC induce A1
expression at least in part by contact-dependent mechanisms, which may
involve the ligation of cell adhesion receptors. Role of adhesion molecules in monocyte stimulation of A1 mRNA expression in EC
We therefore investigated whether the ß2
integrin/ICAM-1 adhesion pathway was important for the increase in A1
expression by EC induced after coculture with monocytes. FACS analysis
of serum-starved EC confirmed that these cells expressed
low/intermediate levels of ICAM-1, but undetectable levels of VCAM-1
and E-selectin (data not shown), a pattern similar to that in
noncytokine-stimulated EC. Monocyte adherence to endothelium is
mediated partly by ß2 integrins, and our initial studies
confirmed that mAbs directed at either the M-chain
(CD11b) or the ß2-chain (CD18) were effective in blocking at least
50% of adhesion of monocytes to serum-starved EC (data not shown).
When these same mAbs were included in EC/monocyte cocultures, however,
there was no alteration in A1 mRNA, compared with control cocultures
incubated with isotype-matched IgG (Fig. 5A). In contrast,
anti-CD31 (PECAM-1) reduced A1 expression in EC/monocyte cocultures
by 50 ± 10% at 21 h (Fig. 5B). Anti-CD31 had no
effect on basal levels of A1 mRNA in EC cultures (data not shown).
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TNF is produced by monocytes and has been reported to cause
up-regulation of A1 gene expression in EC (13). To determine a possible
contribution of TNF to the induction of A1 in EC following coculture
with monocytes, we included anti-TNF mAb at the start of coculture.
Fig. 6 shows that the inclusion of
anti-TNF mAb to EC/monocyte cocultures produced an almost complete
inhibition of the A1 response at 6 h. At 21 h of coculture,
however, anti-TNF mAb only achieved a modest reduction of 32.2
± 15%. Interestingly, the inclusion of anti-TNF mAb completely
abolished any increase in A1 expression when monocytes were separated
from EC by filters (data not shown), suggesting that monocyte-derived
TNF may be responsible for the soluble component of the A1 response in
EC/monocyte cocultures. In contrast, anti-IL-1 mAb did not affect
A1 mRNA expression either in EC/monocyte cocultures or when monocytes
were cultured on filters (data not shown).
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Effect of IL-10 on monocyte-induced A1 expression in EC
Stimulation of TNF synthesis in monocytes is followed by the
induction of IL-10, which acts in an autocrine fashion to inhibit
further TNF production (18), thus limiting the inflammatory response.
When IL-10 was added to EC/monocyte cocultures, no induction of A1
expression was seen at 6 h (Fig. 7A). Hence, IL-10 blocked the
ability of monocytes to increase A1 expression in EC after 6 h of
coculture. The inhibitory effect of IL-10 was less evident when A1
expression was analyzed at 21 h (Fig. 7A). This
provides further support for a role for TNF in the initial induction of
A1 expression by monocytes, while suggesting that non-TNF-dependent
pathways mediate the sustained levels of A1 at later time points.
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B). Thus, IL-10 inhibited A1
induction and also decreased survival of EC cocultured with monocytes. Effect of monocytes on expression of other apoptosis-related genes in EC
bax mRNA was constitutively expressed in EC, and levels
were not altered by serum starvation, coculture with monocytes, or IL-1
stimulation, except for a small increase after serum starvation at +6
h. It is unlikely, however, that changes in bax levels are
responsible for the protective effect of monocytes on survival of
serum-starved EC seen at 21 h because bax expression at
this time was unchanged under all conditions (Fig. 8A).
bcl-xL mRNA was also expressed by unmanipulated
EC, but mRNA levels were not altered by serum starvation, nor by
subsequent coculture with monocytes or by stimulation with IL-1 (Fig. 8B). bcl-2 mRNA was expressed at low levels in EC
and appeared to be up-regulated in response to coculture with
monocytes, but with delayed kinetics compared with A1 (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our experiments suggest that actual cell:cell contact is necessary to generate a signal in EC that leads to the induction of A1, perhaps by the ligation of surface adhesion receptors. We were, however, unable to inhibit the induction of A1 by using mAbs against the CD11b/CD18 complex, despite the fact that these Abs are effective at inhibiting adhesion of monocytes to EC under both normal and serum-starved conditions. PECAM-1 (CD31) is a junctional adhesion molecule that mediates transendothelial migration of leukocytes (19). The reduction in A1 stimulation by anti-PECAM-1 mAbs in EC/monocyte cocultures raises the interesting possibility that monocyte transmigration may be important in stimulating the increase in A1 mRNA in EC. It may be that the engagement of PECAM-1 on transmigrating leukocytes can lead to cell activation (20), which in turn releases signals to the EC to influence cell behavior and survival. Interestingly, the induction of E-selectin gene expression in EC/monocyte cocultures is also inhibited by anti-PECAM-1 mAbs (unpublished observations). It is not yet known whether direct engagement of PECAM-1 on EC results in cell activation.
At least part of the ability of monocytes to induce A1 expression by EC is mediated by TNF, an observation consistent with a previous report that TNF induces A1 mRNA in EC (13). Our results suggest that TNF plays a major role in the early A1 response of EC, but a lesser role at 21 h. Both soluble and membrane-bound TNF may be important; evidence for a role for soluble TNF is provided by the finding that anti-TNF mAb abolished A1 induction when EC were separated from monocytes by 0.45-µm filters (data not shown). Monocyte activation leads initially to the synthesis of inflammatory cytokines such as TNF and IL-1, which then act to stimulate the production of IL-10 (21), an immunomodulatory cytokine with an antiinflammatory role. One action of IL-10 is to prevent further secretion of inflammatory mediators by monocytes (22). Thus, the inhibitory action of IL-10 on A1 expression in EC/monocyte cocultures may be the result of suppression of TNF synthesis by monocytes.
Although we and others observed that TNF increased A1 expression in EC
(13), prolonged exposure to TNF under some circumstances may in fact
induce apoptosis in EC (3). Indeed, TNF induces apoptosis in many cell
types, and many of the intracellular death signals downstream from the
TNF receptor have now been identified (23). EC, however, are not killed
by TNF alone unless coincubated with a protein or mRNA synthesis
inhibitor (24). The antiapoptotic effect of TNF is mediated by the
activation of NF-
B, which, by inducing TNF receptor-associated
factors 1 and 2, and the inhibitors of apoptosis proteins, blocks the
activation of the caspase cascade that leads to death (25). The action
of TNF in inducing A1 expression in EC may represent another
antiapoptotic mechanism. Retroviral-mediated transfer of A1 cDNA to
human microvascular EC protects against death induced either by TNF in
the presence of actinomycin D or by ceramide (24). Therefore, the
increased survival of EC when cultured in the presence of monocytes may
be the result of augmented A1 expression.
A1 contains BH1 and BH2 domains that mediate heterodimerization with the proapoptotic protein bax (26). Thus, A1 may function, like bcl-2 and bcl-xL, by inhibiting bax-induced cytochrome c release from mitochondria, and the subsequent activation of caspase-3. Monocytes had little effect on bax expression at 21 h, when the protective effect on EC was most marked, suggesting it is unlikely that alterations in bax levels influence EC survival in EC/monocyte cocultures. bcl-xL has been shown to protect against ceramide-induced apoptosis in EC. bcl-xL is constitutively expressed by EC, but levels were not significantly altered by coincubation with monocytes. bcl-2 mRNA levels in EC increased in response to coculture with monocytes, but with slower kinetics when compared with A1 mRNA. We cannot, therefore, exclude a possible role for bcl-2 protein in the protection of EC from apoptosis.
Lindner et al. have reported that mononuclear cells preactivated with either LPS or irradiation caused apoptosis of cultured EC after 48-h coincubation, and that this was likely to be mediated by TNF expressed at the monocyte cell surface (27). Lymphocytes present within the mononuclear cell suspensions expressed TGF-ß, which acted on EC to produce cell cycle arrest, even in the absence of prior activation of the leukocytes. In these experiments, IL-10 blocked both TNF-induced apoptosis and TGF-ß-induced cell cycle arrest, confirming an important protective role for this cytokine. While lymphocytes on their own did not stimulate A1 expression in EC, we cannot exclude the possibility that the small number of contaminating lymphocytes may, by interacting with monocytes and/or EC, contribute to the increase in A1 mRNA levels in EC. Lymphocytes adhering to EC may cause the release of platelet-activating factor, which would in turn stimulate the production of TNF. Moreover, the presence of lymphocytes in our monocyte preparations might represent more closely the in vivo situation. The final outcome of TNF stimulation on EC would depend upon the presence of other extracellular signals as well as the activation state of the endothelium itself. In our studies, concomitant stimulation by adherent and/or transmigrating monocytes might initiate signals that would shift the balance toward survival rather than death, while the opposite effect may be produced by preactivated lymphocytes expressing TGF-ß.
The observation that EC receive survival signals in the context of an inflammatory response is important. Previous work has shown that the inflammatory cytokines, TNF and IL-1, are able to induce A1 expression (13), while growth factors and mitogens do not. This points to a role for A1 in protecting EC against death and damage in inflammation. In this study, we have demonstrated that monocytes also increase A1 expression, but, in contrast to the effect seen with cytokines, monocyte-induced A1 expression is sustained for a longer period of time. Vascular endothelium plays a pivotal role in the regulation of inflammatory responses; thus, the maintenance of EC integrity and survival is crucial to the effective development as well as the successful resolution of inflammation. Loss of integrity of EC in uncontrolled inflammation, for example, leads to the capillary leak syndrome, with resulting tissue edema, and the development of the adult respiratory distress syndrome. The elucidation of specific survival signals in the context of inflammation may open up avenues of intervention in the management of conditions in which uncontrolled inflammation leads to endothelial damage and death.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kwee L. Yong, Department of Haematology, University College London, 98 Chenies Mews, London WC1E 6HX, U.K.
3 Abbreviations used in this paper: EC, endothelial cell(s); PECAM, platelet endothelial cell adhesion molecule; PI, propidium iodide.
Received for publication July 2, 1998. Accepted for publication October 23, 1998.
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